Research Project: Integrated Disease Management Strategies for Woody Perennial Species

Location: Crops Pathology and Genetics Research

2018 Annual Report

Objectives
Objective 1: Examine etiology and ecology of key rootstock and scion diseases to enhance sustainability and profitability of tree and vine crops. Subobjective 1A: Conduct transcriptome analysis to identify potential causes of almond bud failure. Subobjective 1B: Determine the epidemiology of Grapevine red blotch-associated virus in California vineyards. Subobjective 1C: Identify potential causes of Paradox canker disease of walnut. Subobjective 1D: Identify soil microbial communities and processes conducive to development of Prunus replant disease. Subobjective 1E: Examine host-induced phenotypic instability in the Sudden Oak Death pathogen Phytophthora ramorum in production nurseries and natural settings. Objective 2: Sequence the genomes of phytoplasmas infecting stone fruit trees in California to enhance development of control and science-based quarantine regulations. Subobjective 2A: Determine the genome sequence of Cherry X disease phytoplasma, Peach yellow leafroll phytoplasma, and Candidatus Phytoplasma pyri. Subobjective 2B: Perform comparative genomics of the Cherry X disease phytoplasma and Peach yellow leafroll phytoplasma with other phytoplasmas. Objective 3: Develop novel amendment-based approaches for the management of soil borne pathogens and diseases. Subobjective 3A: Optimize anaerobic soil disinfestation (ASD) and its effectiveness against key pathogens under in vitro conditions. Subobjective 3B: Enhance and optimize ASD for management of almond orchard replant problems. Subobjective 3C: Characterize microbial community responses to ASD in greenhouse and orchard trials. Subobjective 3D: Quantify greenhouse gas emissions, nitrogen (N)transformations, and inorganic N leachate resulting from ASD. Objective 4: Identify host genotypes that exhibit resistance to key soil borne pathogens. Subobjective 4A: Identify and characterize Juglans rootstock genotypes resistant to Agrobacterium tumefaciens. Subobjective 4B: Identify and characterize Juglans rootstock genotypes resistant to key Phytophthora species. Objective 5: Identify gene and protein targets for use in novel molecular disease management strategies in woody perennial rootstocks. Subobjective 5A: In planta transcriptomic approaches to investigate host-Phytophthora interactions. Subobjective 5B: Examine the feasibility of using RNAi technology to suppress infection by Phytophthora species.

Approach
Objective 1 1A: Collect symptomatic shoots from almond trees exhibiting bud failure (BF) and shoots from non-symptomatic trees. Identify differentially expressed genes in BF trees compared with controls. Validate results of differentially expressed genes to identify BF markers and trees with the genetic potential to exhibit BF. 1B: Monitor grapevines in established plot for the spread of Grapevine red blotch-associated virus (GRBaV). Assess fruit quality of infected grapevines and compare with confirmed non-infected grapevines. Analyze data for variance and spatial and temporal changes in the GRBaV spread. 1C: Examine evidence for host genetic contributions to Paradox Canker Disease (PCD) of walnut. Use established metatranscriptomic libraries to bioinformatically examine signatures of host response to PCD. 1D: Establish plants susceptible to Prunus replant disease (PRD) in replicate plots of soil that induce PRD and replicate plots using the same soil treated so that PRD is not induced. Sample the soil and roots to examine associations of microbial taxa and their activities with PRD induction. 1E: Characterize newly identified plant defense mechanism to explore the feasibility of using nursery ornamentals as a pathosystem. Assess virulence and genetic stability among isolates of P. ramorum. Investigate factors that induce phenotypic instability and reduce aggressiveness towards specific hosts. Objective 2 2A: Purified DNA from petioles of cherry and almond, and the columella of pear fruit will be sheared, barcoded, amplified and sequenced. 2B: Compare annotated genomes to determine quarantine concerns. Examine gene organization by aligning the genomes to visualize regions of synteny and perform other comparative analyses. Objective 3 3A: Perform a series of anaerobic soil disinfestation (ASD) greenhouse trials to screen alternative carbon sources for their ability to generate and maintain anaerobic conditions and for their efficacy in reducing pathogen populations in soil. 3B: Examine efficacy of rice bran and more affordable ASD substrates for control of PRD in a greenhouse soil bioassay. 3C: Characterize microbial community responses to selected ASD carbon sources and organic amendments in the trials described in subobjectives 3 and 3B. 3D: Quantify GHG emissions and nitrate leaching resulting from ASD to facilitate adoption of ASD practices and refine existing biogeochemical models. Objective 4 4A: Produce clonal copies of confirmed interspecific hybrids with resistance to crown gall and Phytophthora. Evaluate clones for resistance. Perform genotyping-by-sequencing (GBS), associated mapping and mapping population analysis. 4B: Produce clonal copies of confirmed interspecific hybrids with resistance to P. cinnamomi and P. citricola. Evaluate clones for resistance. Objective 5 5A: Conduct in planta transcriptomic analyses of P. citricola in walnut and almond. 5B: Select candidate genes from data in subobjective 5A and develop stable host-induced gene silencing lines in walnut.

Progress Report
Sub-objective 1A: Both healthy almond orchards and orchards exhibiting almond bud failure symptoms were identified. Shoot samples were collected from these orchards during late fall and spring for ribonucleic acid (RNA) sequencing work. The non-availability of almond genome in the public domain has become a problem for making progress. To address this, DNA obtained from a shoot sample of almond cultivar, Nonpareil, was subjected to shotgun sequencing using the Illumina sequencing approach. Preliminary bioinformatic analysis conducted in collaboration with a researcher at the University of California, Davis, indicated the depth of coverage was about 35X. Because of the heterozygocity in the almond genome, this data was inadequate and a plan to subject the DNA for sequencing using PacBio system is being implemented. Sub-objective 1B: In 2016, ARS scientists in Davis, California, in collaboration with University of California, Davis researchers, reported successful transmission of Grapevine red blotch virus (GRBV) by Three-cornered alfalfa hopper (Spissistilus festinus, family: Membracidae). Following this, a field trial was set up for studying transmission of GRBV at an experimental farm at the University of California, Davis, field station. Adult insects of S. festinus, fed on grapevines naturally infected with GRBV, were released in cages onto Cabernet Sauvignon grapevines in 2016. These grapevines were examined for GRBV infection in Fall 2017 and Spring 2018 and, so far, do not appear to be infected with the virus. In a commercial Zinfandel vineyard, from a basal level of 25 percent, the incidence of GRBV infection more than doubled in two years. This level of increase is at levels close to what has been observed in a few vineyards in Oregon during a collaborative study with researchers at the Oregon State University (OSU). In several vineyards in California and Oregon, another species of treehopper, (Tortistilus species) has been frequently found. Transmission studies conducted using adults of this species did not detect GRBV transmission. These results are like those reported by OSU collaborators. Sub-objective 1C: To identify potential causes of Paradox canker disease (PCD) of walnut, Illumina sequencing reads of metagenomic DNA and metatranscriptomic RNA isolated from healthy and PCD-affected Paradox rootstocks were processed through bioinformatics pipelines. DNA sequence databases were searched for any matches to known prokaryote, eukaryote, or viral nucleic acid. No pathogen-contig matches were detected. In contrast, when metatranscriptomic RNAs were extracted, sequenced, and joined into contigs from walnut trees known to be infected with Brenerria species (walnut canker pathogen), cherry leaf roll virus (the walnut blackline pathogen), or Phytophthora citricola (walnut crown rot pathogen), sequences of each of the expected pathogens were identified in the metatranscriptomic libraries, which confirms the utility of this approach to identify PCD etiology. Since our initial meta sampling and sequencing from PCD tissues, ARS researchers established graft transmission experiments in efforts to improve chances for pathogen detection in association with PCD. Sub-objective 1D: Metabarcoding of ribosomal ribonucleic acid (rRNA) genes was conducted from bacteria, fungi, and oomycetes isolated from roots and soil collected in a greenhouse bioassay and five almond orchard replant trials. Samples were collected, preplant and postplant, from plots treated to remediate Prunus replant disease (PRD) and from non-treated plots. In two of the orchard trials, samples were collected at four monthly intervals after planting in efforts to effectively capture potential microbial population dynamics that may affect tree responses to preplant treatments in almond replant settings. Sub-objective 1E: Examine host-induced phenotypic instability in the Sudden Oak Death pathogen, Phytophthora ramorum in production nurseries and natural settings. As shown previously, the Sudden Oak Death pathogen, P. ramorum, undergoes genome and phenotypic alterations when it interacts with oak hosts but not with foliar hosts. This phenomenon is a previously undescribed host-pathogen interaction that may facilitate development of novel disease management strategies. To understand the host-dependency of the phenomenon, phenotypes of diverse non-oak isolates taken from Rhododendron foliage and other ornamental plants, as well as from natural host species, soil, and water were analyzed. Isolates recovered from artificially inoculated oak logs were also examined. A correlation between such phenotypes as colony shape and growth rate on culture media with aggressiveness in Rhododendron foliage, was observed. Sub-objective 2A: To determine the genome sequence of Cherry X disease phytoplasma, Peach yellow leafroll phytoplasma, and Candidatus Phytoplasma pyri., a shoot sample of a cherry tree showing Western X disease phytoplasma infection was subjected to shotgun DNA sequencing using the Illumina HiSeq platform. The sequence data is being analyzed for retrieval and assembly of phytoplasma sequences. Efforts have been made to identify other sources of Western X disease phytoplasma isolated in the Pacific West by reaching out to researchers and state agriculture officials in Oregon and Washington where this phytoplasma has been found in recent surveys. Sub-objective 3B: Greenhouse and field trials were conducted to evaluate efficacy of; 1) rice bran and six additional Anaerobic Soil Disinfestation (ASD) substrates (almond hull, almond shell, almond hull plus shell, pistachio hull, tomato pomace, grape pomace (field), olive pomace (field) and mustard seed meal), 2) soil pasteurization, and 3) soil fumigation was completed using two almond replant soils. Efficacy of each of the treatments was evaluated according to growth responses of Nemaguard peach seedlings growing in the treatments cited above. Rice bran was the most consistently effective ASD substrate; however, other less expensive ASD substrates such as almond hull plus shell mixture provided adequate Prunus Replant Disease control and may be more economical, compared to rice bran. This will greatly enhance grower acceptability of this form of soil fumigation. Sub-objective 3C: Metabarcoding of rRNA genes was performed from bacteria, fungi, and oomycetes collected from five almond orchard replant trials, including ASD treatments with rice bran, almond hull and shell mixture; preplant soil fumigation treatments; and non-treated controls. Samples were collected from roots and/or soil before planting and at one or more intervals after planting, from plots treated to remediate prunus replant disease (PRD) and from non-treated plots. Sub-objective 4A: Approximately 200 members of the mapping population which resulted from a cross between, Juglans microcarpa and Juglans regia ‘Serr’ were screened for resistance to crown gall caused by the bacterium, Agrobacterium tumefaciens. Crown gall severity ranged from resistant to highly susceptible indicating that the breeding population was segregating for disease resistance. These data were used to map the genetic locus which mediates crown gall resistance to chromosome 11 in J. microcarpa. Sub-objective 4B: Greenhouse experiments were conducted to screen 495 clonal genotypes of Juglans microcarpa x Juglans regia ‘Serr’ hybrids for their resistance to Phytophthora cinnamomi and P. citricola. The experiments identified multiple clonal selections that expressed resistance to the pathogens and facilitated mapping of the resistance to chromosome 11 in the J. microcarpa genome. Sub-objective 5A: The genetic mechanisms underlying infection and disease development in Phytophthora root and crown rots of almond and walnut are poorly understood. Proteins were isolated from disease lesions on almond seedlings caused by Phytophthora citricola as well as tissues of non-infected plants. Proteomic analysis showed that most of these proteins belonged to almond, but 71 proteins were derived from the pathogen. The pathogen proteins were highly expressed inside the almond tissue and are involved in detoxification and respiration. This sheds light on the pathogenesis mechanisms which may be exploited in the development of disease management strategies. Sub-objective 5B: This project is designed to engineer rootstocks of walnut and almond to confer host-induced gene silencing (HIGS)-based resistant to Phytophthora. The first step consisted of identifying 300 Phytophthora genes that were conserved among genomes of five diverse Phytophthora pathogens, but their homologous sequences do not exist in plant or fungal genomes. Transcriptome data of these 300 P. citricola genes revealed approximately 60 genes which are active in both saprobic and pathogenic phases. From this group, 24 candidate genes were selected and used in tests to develop RNAi-based disease suppression.

Accomplishments
1. Characterization of soil microbial communities responsive to anaerobic soil disinfestation (ASD), an alternative to chemical-based soil fumigation. ARS researchers in Davis, California, identified microbial community shifts, as a function of ASD carbon source, which are associated with suppression or killing of plant pathogens. Results from replicated greenhouse and field trials indicate rice bran, and the more cost-effective tomato pomace, stimulate proliferation of bacteria involved in the nitrogen cycle (i.e., nitrogen-fixation and denitrification) and produce volatile organic compounds known to be inhibitory to plant pathogens. Both carbon sources are equally effective at reducing populations of target plant pathogens under greenhouse and field conditions. ASD using tomato pomace may be a cost-effective alternative to the use of chemical soil fumigants known to be harmful to human health and the environment.

2. Identification and mapping of resistance to Phytophthora species among hybrid walnut rootstock genotypes. ARS researchers in Davis, California, working in collaboration with colleagues at the University of California, Davis, identified walnut rootstock clones with resistance to Phytophthora cinnamomi and P. citricola and mapped this resistance to a region of chromosome 11 in Juglans microcarpa. A breeding population, comprised of hundreds of clonal genotypes from controlled crosses between J. microcarpa and J. regia ‘Serr’, was characterized for its resistance to both pathogens and then statistically evaluated for the relationship of resistance to genetic markers in the walnut genome. Linkage of Phytophthora resistance with markers on chromosome 11 is the first genetic localization of resistance to the pathogen in walnut. These results are being used to facilitate development of marker-assisted breeding strategies for resistance to the pathogen in walnut rootstocks.

3. Identifying and mapping genetic loci which mediate resistance to Agrobacterium tumefaciens in walnut (Juglans) genotypes. ARS researchers in Davis, California, working in collaboration with colleagues at the University of California, Davis, identified multiple walnut rootstock genotypes with resistance to Agrobacterium tumefaciens, the causative agent of crown gall. A breeding population comprised of 600 progenies from controlled crosses between Juglans microcarpa and J. regia ‘Serr’ was screened for resistance to A. tumefaciens. Using a method known as quantitative trait locus loci (QTL) analysis, resistance to A. tumefaciens was mapped to a region of chromosome 11 in J. microcarpa. Selections of the resistant clones have been advanced to commercial propagation and large-scale field evaluations for resistance. The linkage of crown gall resistance with genetic markers on chromosome 11 is the first genetic localization of resistance to a pathogen in walnut.

4. Phenotypic diversification in Phytophthora ramorum. A single clonal lineage of P. ramorum dominates forests of the Pacific Northwest and causes Sudden Oak Death. However, even though all isolates are genetically indistinguishable, variations in growth rate and aggressiveness allowed ARS researchers in Davis, California, to place isolates into three distinctive groups. Group 1 and group 2 isolates were indistinguishable by their growth rates on an artificial medium; however, group 1 isolates were significantly more aggressive in plants than group 2 isolates. Group 3 isolates were less aggressive than group 1 isolates and their colony morphology differed from group 1 and group 2 isolates. All three groups have been found in production nurseries, which demonstrates that less aggressive individuals have selective advantage in certain conditions over more aggressive isolates thereby influencing development of disease management strategies and subsequent regulatory restrictions.

5. Cherry leafroll virus can infect black walnut species. Black walnuts (Juglans hindsii, J. major, J. nigra) are believed to be resistant to Cherry leafroll virus (CLRV) that causes blackline disease of English walnut (J. regia) trees grafted on black walnut derived rootstocks. Using molecular approaches, ARS scientists in Davis, California, discovered that several asymptomatic black walnut accessions at the USDA ARS National Clonal Germplasm Repository in Davis, California, were infected by CLRV. This indicates the occurrence of resistance-breaking CLRV isolates. The use of these avirulent CLRV isolates are being utilized for the prevention of blackline disease of walnuts using a cross-protection-based approach. In addition, several J. regia accessions were found free of CLRV and are now being used as a source of recessive host alleles for virus resistance.

6. All morphology types of the treehopper, Tortistilus species, in a California vineyard belong to a single species. To find potential treehopper insect vectors of Grapevine red blotch virus, it was found that four types of treehoppers; i.e., brown horned, green horned, brown non-horned and green non-horned treehoppers were feeding on grapevines in vineyard blocks exhibiting red blotch disease. It has been difficult to use these treehoppers collected from vineyards for transmission experiments because of the inability to identify them due to their contrasting morphological traits. However, ARS scientists in Davis, California, now know that all four types belong to the same treehopper species as revealed by shotgun DNA sequencing. It is now possible to bar code their DNA and use these insects in virus transmission studies.

Review Publications
Knipfer, T., Barrios-Masias, F., Cuneo, I., Bouda, M., Albuquerque, C., Brodersen, C., Kluepfel, D.A., McElrone, A.J. 2018. Variations in xylem embolism susceptibility under drought between intact saplings of three walnut species. Tree Physiology. 38(8):1180-1192. https://doi.org/10.1093/treephys/tpy049.
McCartney, M., Roubtsova, T., Yamaguchi, M., Kasuga, T., Ebeler, S., Davis, C., Bostock, R. 2017. Effects of Phytophthora ramorum on volatile organic compound emissions of Rhododendron using gas chromatography-mass spectrometry. Analytical and Bioanalytical Chemistry. 410(5):1475-1487. https://doi.org/10.1007/s00216-017-0789-5.
Hao, W., Miles, T.D., Martin, F.N., Browne, G.T., Forster, H., Adaskaveg, J.E. 2018. Temporal occurrence and niche preferences of Phytophthora spp. causing brown rot of citrus in the Central Valley of California. Phytopathology. 108(3):384-391. https://doi.org/10.1094/PHYTO-09-17-0315-R.
Browne, G.T., Ott, N.J., Poret-Peterson, A.T., Gouran, H., Lampinen, B.D. 2018. Efficacy of anaerobic soil disinfestation for control of Prunus replant disease. Plant Disease. 102:209-218. https://doi.org/10.1094/PDIS-09-16-1392-RE.
Stevens, K.A., Woeste, K., Chakraborty, S., Crepeau, M.W., Leslie, C.A., Martinez-Garcia, P.J., Puiu, D., Romero-Severson, J., Coggeshall, M., Dandekar, A.M., Kluepfel, D.A., Neale, D.B., Salzberg, S.L., Langley, C.H. 2018. Genomic variation among and within six Juglans species. G3, Genes/Genomes/Genetics. 8(7):2153-2165. https://doi.org/10.1534/g3.118.200030.
Baumgartner, K., Fujiyoshi, P.T., Ledbetter, C.A., Duncan, R., Kluepfel, D.A. 2018. Screening almond rootstocks for sources of resistance to Armillaria root disease. HortScience. 53(1):4-8. https://doi.org/10.21273/HORTSCI12038-17.
Elliot, M., Yuzon, J., Malar, M.C., Tripathy, S., Bui, M., Chastagner, G.A., Coats, K., Rizzo, D.M., Garbelotto, M., Kasuga, T. 2018. Characterization of phenotypic variation and genome aberrations observed among Phytophthora ramorum isolates from diverse hosts. BMC Genomics. 19:320. https://doi.org/10.1186/s12864-018-4709-7.
Poret-Peterson, A.T., Bhatnagar, S., McClean, A.E., Kluepfel, D.A. 2017. Draft genome sequence of agrobacterium tumefaciens biovar 1 strain 186, isolated from walnut. Genome Announcements. 5(46):e01232-17. https://doi.org/10.1128/genomeA.01232-17.
Preto, C.R., Sudarshana, M.R., Zalom, F.G. 2018. Feeding and reproductive hosts of Spissistilus festinus (Say) (Hemiptera: Membracidae) found in Californian vineyards. Journal of Economic Entomology. 111(6), 2531-2535. https://doi.org/10.1093/jee/toy236.